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Abstract #15142 Published in IGR 8-4
Contractility as a prerequisite for TGF-β-induced myofibroblast transdifferentiation in human tenon fibroblasts
Meyer-Ter-Vehn T; Sieprath S; Katzenberger B; Gebhardt S; Grehn F; Schlunck GInvestigative Ophthalmology and Visual Science 2006; 47: 4895-4904
PURPOSE: To assess the significance of Rho-kinase-dependent contractility in TGF-β-induced myofibroblast transdifferentiation of human tenon fibroblasts to characterize possible pharmacological targets for the inhibition of postoperative scarring after glaucoma surgery. METHODS: Human tenon fibroblasts (HTFs) were grown in culture and stimulated with TGF-β1. The effect of TGF-β on Rho-GTPase activity was assessed by GST-rhotekin binding domain pulldown assay and detected by Western blot analysis. Contractility was evaluated in a silicone substrate wrinkling assay and in fibroblast-populated collagen gels. The actin cytoskeleton and focal adhesions were visualized by immunofluorescence microscopy. alpha-SMA transcripts were measured by real-time RT-PCR. TGF-β-induced Smad- and p38-activation and expression of alpha-SMA were detected by Western blot analysis. Nuclear translocation of Smad2/3 was determined by confocal immunofluorescence microscopy. The influence of Rho-dependent kinase (ROCK) and myosin light chain kinase (MLCK) were studied by using specific kinase inhibitors (Y-27632, HA-1077, H-1152, and ML-7). RESULTS: Within 10 minutes of stimulation, TGF-β induced Rho activation that was associated with an increase in cell tension and followed by actin stress fiber enhancement. ROCK inhibitors released cell tension and averted TGF-β-induced cytoskeletal changes, p38 activation and subsequent alpha-SMA expression, whereas Smad2-phosphorylation and nuclear translocation were preserved. MLCK inhibition also blocked alpha-SMA expression. In fibroblast-populated collagen lattices, ROCK inhibitors prevented TGF-β-induced stress fiber assembly and contraction. CONCLUSIONS: TGF-β induces a rapid contractile response in HTFs that precedes myofibroblast transdifferentiation. ROCK inhibitors release this contraction and block subsequent TGF-β-induced myofibroblast transdifferentiation and may therefore serve to modulate postoperative scarring after glaucoma filtering surgery.
Dr. T. Meyer-ter-Vehn, Division of Experimental Ophthalmology and Glaucoma Center, Würzburg University Eye Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg, Germany
Classification:
3.8 Pharmacology (Part of: 3 Laboratory methods)
2.3 Sclera (Part of: 2 Anatomical structures in glaucoma)
12.8.10 Woundhealing antifibrosis (Part of: 12 Surgical treatment > 12.8 Filtering surgery)
